Sustained hyperglycemia is associated with the development of diabetic nephropathy. The goal of this project is to distinguish the direct toxicity of hyperglycemia from indirect actions mediated through the formation of glycated proteins, alpha-dicarbonyl intermediates and Advanced Glycation End Products (AGEs) on proteins (glycooxidation products). Glucose and reactive carbonyls induce direct stress through generation of reactive intermediates and auto-oxidation. In contrast glycated proteins and AGEs induce both direct oxidative stress and indirect cellular oxidative stress mediated through specific responses. The central hypothesis to be tested is that the induction of intracellular oxidant stress represents a major final common pathway by which both direct and indirect effects of hyperglycemia induce changes leading to diabetic nephropathy. We propose: SA#1: To identify the causes of oxidative stress in the diabetic kidney in rats, focusing on histochemical markers, and mechanisms of transition from early to late nephropathy. Early renal changes caused by diabetes are typified by tubular and glomerular hypertrophy late lesions by interstitial fibrosis and glomerular sclerosis. We plan to characterize the transitional, with emphasis on the role of tubular injury in initiating irreversible renal damage. SA#2: To test the hypothesis that tubular cells in culture are more susceptible than mesangial cells to oxidative stress induced directly by hyperglycemia and indirectly by AGEs and other glycooxidation products. Because the renal tubule plays a key role in the processing and excretion of protein- associated and free AGEs, tubular cells may be uniquely susceptible to the induction of oxidative stress, as indicated by our preliminary data. SA#3: To test the validity and excretion of free AGEs, the lipid peroxidation product isoprostane, and an enzyme marker of tubular damage as predictors of the progression of diabetic nephropathy. This aim will integrate in vitro results with human disease outcomes, by identifying predictors that can serve as surrogate markers for nephropathy in clinical trials for antioxidant therapies.